Animal care
All animal experiment was designed and conducted in accordance with the Guide of the Care and Use of Laboratory Animals, the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research, and approved by the Institutional Animal Care and Use Committee for Soonchunhyang University Hospital Bucheon.
Mice used in this study were C57/BL6 strain (8 weeks, male, 22 ~ 24 g) and purchased from the Orient Bio Inc., (Seongnam, South Korea). All mice were housed in breeding cages in a room with a 12/12-hour light/dark cycle and had free access to food and water. The humidity and temperature were respectively maintained at 50% and 23 ~ 26℃.
Induction Of Diabetes
Diabetes was induced by a single i.p. injection of STZ (150 mg/kg; Sigma-Aldrich, St. Louis, MO, USA). STZ was freshly prepared in 100 mM citrate buffer (pH 4.5). To avoid sudden hypoglycemia post-injection, the mice were fed 10% sucrose overnight. Hyperglycemia was confirmed by measuring blood glucose using the Accu-Check active blood glucose monitor (Roche Diagnostics, Mannheim, Germany) 2 days and 2 weeks post-injection. Only mice with non-fasting blood glucose levels > 300 mg/dL, polyuria, or glucosuria were defined as STZ-induced diabetic mice and used in the experiments. Non-diabetic, age-matched mice were used as the controls. The general parameters of mice obtained on the day of the sacrifice are given in Table 1.
Grouping And Treatment Regimen
To evaluate the natural course of DR, we observed 60 mice over 6 months of period. Animals were assigned to 6 groups depending on observation period: 1) Normal control, 2) 1 m DM, 3) 2 m DM, 4) 3 m DM, 5) 4 m DM and 6) 6 m DM (Fig. 2a). The effects of short-term hyperglycemia on the retina were investigated on 40 mice, divided into 4 groups: 1) Normal control, 2) 1 m DM, 3) 1 m DM/PHL 4) 1 m DM/Rapa (Fig. 2b). Mice in the 1 m DM/PHL group received two daily subcutaneous injections of phlorizin (Cayman Chemical, Ann Arbor, MI) dissolved in 60% propylene glycol in phosphate-buffered saline at a dose of 200 mg/kg of body weight during the last 7 full days of the experiment and the morning of the 8th day, 3 h before sacrifice. Mice in the 1 m DM/Rapa group, received daily i.p. injections of rapamycin (Sigma-Aldrich, St. Louis, MO) diluted in 4% ethanol and 5% Tween-20 in distilled water at a dose of 3 mg/kg for the same period as for the 1 m DM/PHL group [43].
The effects of long-term hyperglycemia on the retina of diabetic mice were evaluated on 40 mice, divided into 4 groups: 1) Normal control, 2) 3 m DM, 3) 3 m DM/PHL, and 4) 3 m DM/MHY (Fig. 2c). Mice in the 3 m DM/PHL group received two daily subcutaneous injections of PHL during the last 10 full days of the experiment and the morning of the 11th day, 3 h before sacrifice. Mice in the 3mDM/MHY group received daily i.p. injections of MHY1485 (MedChem Express, Monmouth Junction, NJ) at a dose of 10 mg/kg for the same period as for the 3 m DM/PHL group. Eight diabetic mice died in the course of this study.
Tissue Collection And Preparation
After completing the experiment, mice were used for WB and histologic analysis. For histologic analysis, mice were deeply anesthetized and perfused intracardially with 0.1M phosphate buffer (PB) containing 1000 U/ml of heparin, followed by an infusion of 4% paraformaldehyde (PFA) in 0.1M PB. The eyecup was made by removing the anterior segment from the enucleated mouse eye and fixed with 4% paraformaldehyde (Biosesang, Seongnam, South Korea) for 1 h, dehydrated in 30% sucrose overnight, and embedded in frozen section compound (Leica Biosystems Richmond, IL). The sample collection for the WB experiment was that the neuroretina was separated from the enucleated mouse eyes, pooled together and stored at − 80 °C until the experiment.
Western Blot
Neuroretinal tissue was disrupted in RIPA II lysis buffer (Gendepot, Barker, Tx) containing Xpert phosphatase inhibitor cocktail (Gendepot, Barker, Tx) and Xpert protease inhibitor cocktail (Gendepot, Barker, Tx) for 1 h on the ice. Insoluble material was removed by centrifugation (13000 rpm, 15 min, 4 °C), and the only supernatant was obtained. Protein lysate was quantified using Pierce BCA Protein Assay kit (Thermo scientific, Middlesex, MA) according to the manufacturer’s instruction.
The lysates were denatured in 4x Laemmli sample buffer (Gendepot, Barker, Tx), boiled for 10 min at 95 °C. Aliquots of each sample with an equal amount of protein were separated using SDS-acrylamide gel in the range from 6 to 10%, depending on the molecular weight of the target antibody and transferred onto polyvinylidene fluoride membrane (ATTO, Amherst, NY). The membranes were incubated with 5% skim milk in Phosphate-Buffered Saline (PBS) containing 0.1% Tween-20 for detection of total proteins and 5% BSA in PBS containing 0.1% Tween-20 for phosphorylated proteins for 1 h at room temperature and incubated with anti-mTOR, anti-phospho-S6 ribosomal protein (S240/244) from Cell signaling technology (Danvers, MA), anti-phospho-mTOR (S2448), anti-GLUT1 from Abcam (Cambridge, UK), anti-β-actin from Santa Cruz Biotechnology (Dallas, TX) antibodies overnight at 4 °C; details on antibodies are given in Supplemental Table S1. After washing, the membranes were incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG and goat anti-mouse IgG (Genedepot, Barker, Tx) at room temperature for 2 h. The immunoreactive signal was developed using Western blotting detection kit (EzWestLumi plus, ATTO Corporation, Tokyo, Japan) and read with Azure Biosystems™ c280 (Azure Biosystems, Dublin, CA, USA). Bands on blots were quantified using the ImageJ software (National Institutes of Health, Bethesda, MD).
Immunofluorescence
Fresh eyecup cryosections (10 µm in thickness) were made with a Cryotome (Thermo Fisher Scientific Shandon Cryotome, Middlesex, MA). The slides were washed with 1xPBS containing 0.1% Triton X-100 (Sigma-Aldrich, St. Louis, MO) and blocked with 5% donkey serum in PBST for 1 h. The blocked slides were then incubated with anti-phospho-mTOR (S2448), anti-GLUT1 from Abcam (Cambridge, UK), anti-phospho-S6 ribosomal protein (S240/244) and anti-GFAP (CST) from Cell signaling technology (Danvers, MA), anti-Glutamine synthetase from Millipore (Billerica, MA), anti-NeuN from Millipore (Billerica, MA), anti Calbindin from Sigma-Aldrich (St. Louis, MO), anti-Cleaved caspase-3 from Cell signaling technology (Danvers, MA), anti-Beclin1 from Novus Biologicals (Littleton, CO) antibodies for 2 h and detected with secondary antibodies (Alexa Fluor 488 or 568 or 647, dilution 1:1000; Invitrogen Corp., Carlsbad, CA); details on antibodies are given in Supplemental Table S1. Nuclear counterstaining was performed with Hoechst 33342 (Invitrogen Corp, Carlsbad, CA). After washing, the sections were mounted with fluorescence mounting medium (Dako, Santa Clara, CA).
Image Analysis And Cell Counting
All slides were imaged using a Leica SP8 (Leica Microsystems, Wetzlar, Germany) confocal microscope. Cleaved caspase-3, NeuN and Hoechst positive cells (n = 6 per group) were manually calculated using Image J software (National Institutes of Health, Bethesda, MD).
String Analysis
The potential interactions among selected proteins were analyzed using free online database of currently known proteins – STRING (Search Tool for the Retrieval of Interacting Genes, v11.0). IDs of selected proteins were used as an input in the database (http://string-db.org/) to find functional associations in homo sapiens and mus musculus with a minimum interaction score of 0.400 (medium confidence). Active interaction sources included text mining, experiments, databases, gene fusion, co-occurrence, co-expression, and neighborhood.
Statistical analysis
Data are expressed as means ± standard deviation. Differences among groups were analyzed using the Mann-Whitney U test. Differences were considered statistically significant when P ≤ 0.05.